Cells on glass coverslips were analyzed by sequential scanning confocal fluorescent microscopy with a PL APO 63×1.4 oil objective (TSP SP, Leica, Deerfield, IL, USA or LSM 510, Zeiss, Göttingen, Germany [22], [40]. The images were combined by using PhotoShop (Mountain View, CA, USA) and analyzed with the ImageJ software.
Images were acquired by sequential scanning of the immunostained cells with an Olympus Fluorescence Microscope (Olympus, FluoView FV1000) at laser lines of 405, 488, and 594 nm based on the specific Alexa-fluor secondary antibody tag used.
Z-stack images of fluorescent lung sections were acquired by sequential scanning every 1 µm and taken at a 2,048 × 2,048 pixel size, with a line average of 2 to reduce noise.
The slides were evaluated by sequential scanning using a confocal laser scanning microscope (TCS SP5, Leica, Bensheim, Germany).
High magnification images were acquired using a 63× HCX PL APO (with 4 digital zoom factor) oil immersion objective (numerical aperture 1.32) by sequential scanning to minimize the crosstalk of fluorophores.
Confocal images were acquired by sequential scanning using a two-frame Kalman filter and a z-separation of 1 µm.
Triple stained images were obtained by sequential scanning for each channel to eliminate the crosstalk of chromophores and to ensure reliable quantification of co-localization.
For co-localization studies, channels were acquired by sequential scanning.
Confocal sections were obtained with a Leica TCS SP2 AOBS confocal laser microscope (Leica Microsystems S.r.l., Milano, Italy) by sequential scanning.
